Bioanalytical techniques commonly require reagents, buffers, and equipment that is free of contaminating DNA. Contaminating DNA can interfere with high-sensitivity methods, such as hybrid assay, and can imperil any process that relies upon DNA amplification prior to subsequent analysis, e.g., sequencing. Contaminating DNA can originate from a researcher directly or it can be introduced by way of contaminated surfaces, reagents, buffers, or air. Accordingly, detailed protocols are available for creating “DNA-free workspaces” and for clearing buffers, etc. from contaminating DNA.
A common protocol for providing a “DNA-free workspace” relies on regular bleach washing of all laboratory surfaces and equipment, the use of absorbent bench pads on all surfaces, and the use of dedicated hood space for certain key procedures (e.g., PCR). Other protocols recommend regularly exposing surfaces to UV light in order to degrade or inactivate contaminating DNA. Maintaining a “DNA-free workspace” also involves judicious use of disposable labware, such as pipette tips and sample vials.
In addition to keeping a clean workspace, analytical reagents, washes, and buffers must be free of contaminant DNA. In many cases, this involves procuring “DNA-free” reagents or buffers from manufacturers who prepare the reagents and buffers under strict conditions and perform post-production analysis to assure that no DNA is present. For example DNA-free buffers are available from MoBio (Carlsbad, Calif.). The cost of using certified DNA-free reagents can be substantial, however. For example, 500 ml of DNA-free PBS buffer may cost approximately $50 with shipping.
In other situations, DNA contamination may be removed from reagents, buffers, and samples using DNase and DNase clean-up kits. DNases are endonucleases that catalyze hydrolytic cleavage of phosphodiester linkages in the DNA backbone. A variety of DNases are known, and they may cleave DNA in different places (e.g., ends, mid-chain, specific sequences), or cleave single-stranded DNA over double-stranded DNA, or vice versa. A DNase treatment of a reagent will typically involve introduction of prepared DNase, such as AMBION DNase I (Life Technologies, Carlsbad, Calif.) along with a buffered solution containing substrates and optimized ionic species. In some protocols, it may be necessary to use high-turnover, recombinant DNase, such as TURBO DNase, also available from Life Technologies. After a reagent or sample is treated with DNase, the DNase may be degraded with heat, alcohol, or EDTA in order to prevent interference by the DNase in subsequent processing.
While good laboratory practices and judicious use of DNase can prevent much DNA contamination, some instances of DNA contamination are harder to control. For example, precision instrument components, such as ports, injectors, and columns are not typically disposable because of the high manufacturing costs. Additionally, it may not be possible to easily decontaminate the components using standard techniques (e.g., bleach cleaning) because the cleaning compounds may damage the components. In other situations where direct decontamination is not possible, it may also not be feasible to use DNase to digest contaminant DNA because of concerns over cross-contamination or an inability to deactivate the DNase afterward with heat or alcohol. DNase degradation products may also become a source of contamination in proteomic measurements.
Additionally, reliance on certified DNA-free reagents and disposable labware is expensive and produces a large amount of solid waste.